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• W2003702834 abstract "Poxviruses are large DNA viruses that include the causal agent of human smallpox and vaccinia virus. Poxviruses replicate in cytoplasmic foci known as DNA factories. Here we show that a virus-encoded transcription factor, viral mRNA, cellular RNA-binding protein heterodimer G3BP/Caprin-1 (p137), translation initiation factors eIF4E and eIF4G, and ribosomal proteins are concentrated in the same subdomains of cytoplasmic DNA factories. Furthermore, a cell coinfected with two recombinant vaccinia viruses expressing a virus core protein fused to cyan or yellow fluorescent protein displayed separate cyan and yellow factories, indicating that each factory formed from a single genome and was the site of transcription and translation as well as DNA replication. Hijacking of the host translation apparatus within the factory likely enhances the efficiency of virus replication and contributes to the suppression of host protein synthesis, thereby facilitating poxvirus subjugation of the cell. Poxviruses are large DNA viruses that include the causal agent of human smallpox and vaccinia virus. Poxviruses replicate in cytoplasmic foci known as DNA factories. Here we show that a virus-encoded transcription factor, viral mRNA, cellular RNA-binding protein heterodimer G3BP/Caprin-1 (p137), translation initiation factors eIF4E and eIF4G, and ribosomal proteins are concentrated in the same subdomains of cytoplasmic DNA factories. Furthermore, a cell coinfected with two recombinant vaccinia viruses expressing a virus core protein fused to cyan or yellow fluorescent protein displayed separate cyan and yellow factories, indicating that each factory formed from a single genome and was the site of transcription and translation as well as DNA replication. Hijacking of the host translation apparatus within the factory likely enhances the efficiency of virus replication and contributes to the suppression of host protein synthesis, thereby facilitating poxvirus subjugation of the cell. Poxviruses are large DNA viruses that cause human smallpox and molluscum contagiosum in addition to several zoonoses. They are distinguished from other DNA viruses by replicating exclusively in the cytoplasm (Moss, 2007Moss B. Poxviridae: The viruses and their replicaton.in: Knipe D.M. Fields Virology. Lippincott Williams & Wilkins, Philadelphia2007: 2905-2946Google Scholar). Most of what we know about poxvirus replication derives from studies of vaccinia virus (VACV), which was used as the vaccine to eradicate smallpox, a disease caused by a closely related family member. VACV contains nearly 200 genes, which encode proteins that function in disabling host defenses, enabling replication and transcription of the viral genome, and assembling virus particles. The genes can be divided into early, intermediate, and late classes based on their time of expression. The multisubunit viral DNA-dependent RNA polymerase and factors needed for the early stage of transcription are packaged within the infectious particle, allowing synthesis of mRNA soon after entry into the cytoplasm, whereas intermediate and late transcription require de novo viral protein synthesis and genome replication. Genome replication and assembly of virus particles occur in cytoplasmic domains often referred to as DNA factories (Cairns, 1960Cairns J. The initiation of vaccinia infection.Virology. 1960; 11: 603-623Crossref PubMed Scopus (99) Google Scholar, Dales and Siminovitch, 1961Dales S. Siminovitch L. The development of vaccinia virus in Earle's L strain cells as examined by electron microscopy.J. Biophys. Biochem. Cytol. 1961; 10: 475-503Crossref PubMed Scopus (153) Google Scholar, Harford et al., 1966Harford C. Hamlin A. Riders E. Electron microscopic autoradiography of DNA synthesis in cell infected with vaccinia virus.Exp. Cell Res. 1966; 42: 50-57Crossref PubMed Scopus (14) Google Scholar). Early after infection (<4 hr), many of the DNA factories are bounded by rough endoplasmic reticulum, which becomes dispersed at the start of intermediate or late viral protein synthesis and virus assembly (Tolonen et al., 2001Tolonen N. Doglio L. Schleich S. Locker J.K. Vaccinia virus DNA replication occurs in endoplasmic reticulum- enclosed cytoplasmic mini-nuclei.Mol. Biol. Cell. 2001; 12: 2031-2046Crossref PubMed Scopus (142) Google Scholar). Poxviruses employ a variety of specific and global mechanisms to evade host defenses. The former include the synthesis of viral proteins that target mediators of innate immunity such as interferons, tumor necrosis factors, interleukins, complement, and chemokines as well as intracellular signal transduction pathways (Seet et al., 2003Seet B.T. Johnston J.B. Brunetti C.R. Barrett J.W. Everett H. Cameron C. Sypula J. Nazarian S.H. Lucas A. McFadden G. Poxviruses and immune evasion.Annu. Rev. Immunol. 2003; 21: 377-423Crossref PubMed Scopus (465) Google Scholar). On a broader level, poxviruses downregulate the synthesis of many cellular gene products at intermediate and late times. Thus, microarray analyses indicate substantial decreases of most host mRNAs (Brum et al., 2003Brum L.M. Lopez M.C. Varela J.C. Baker H.V. Moyer R.W. Microarray analysis of A549 cells infected with rabbitpox virus (RPV): A comparison of wild-type RPV and RPV deleted for the host range gene, SPI-1.Virology. 2003; 315: 322-334Crossref PubMed Scopus (37) Google Scholar, Guerra et al., 2003Guerra S. Lopez-Fernandez L.A. Pascual-Montano A. Munoz M. Harshman K. Esteban M. Cellular gene expression survey of vaccinia virus infection of human HeLa cells.J. Virol. 2003; 77: 6493-6506Crossref PubMed Scopus (98) Google Scholar), consistent with earlier data (Rice and Roberts, 1983Rice A.P. Roberts B.E. Vaccinia virus induces cellular mRNA degradation.J. Virol. 1983; 47: 529-539Crossref PubMed Google Scholar). Rapid degradation of cellular and viral mRNAs is initiated by a recently discovered poxvirus-encoded decapping enzyme (Parrish et al., 2007Parrish S. Resch W. Moss B. Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression.Proc. Natl. Acad. Sci. USA. 2007; 104: 2139-2144Crossref PubMed Scopus (80) Google Scholar, Shors et al., 1999Shors T. Keck J.G. Moss B. Down regulation of gene expression by the vaccinia virus D10 protein.J. Virol. 1999; 73: 791-796PubMed Google Scholar). Augmented translation of viral mRNAs has been suggested from in vitro studies, though a structural explanation of this has not been determined (Bablanian et al., 1991Bablanian R. Goswami S.K. Esteban M. Banerjee A.K. Merrick W.C. Mechanism of selective translation of vaccinia virus mRNAs: Differential role of poly(A) and initiation factors in the translation of viral and cellular mRNAs.J. Virol. 1991; 65: 4449-4460Crossref PubMed Google Scholar). In the current study, we provide a mechanism for preferential expression of viral genes based on compartmentalization of viral mRNA and transcription factors with cellular translation factors within the virus factory. Furthermore, evidence for viral transcription and translation within the factory was obtained by simultaneously infecting cells with two recombinant poxviruses; individual factories within a cell contained unique protein products of each recombinant, demonstrating the origin of a factory from a single viral genome and the linkage of transcription and translation sites. Hijacking elements of the translation apparatus allows the coordination of viral mRNA and protein synthesis at sites of virus assembly and can contribute to the suppression of cellular protein synthesis, facilitating virus takeover and global obstruction of host responses. Viral DNA factories are commonly visualized by fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) or Hoechst stain as one or more juxtanuclear structures. Acridine orange, in contrast to these DNA-specific fluorochromes, can be used to visualize RNA as well as DNA (Zelenin, 1999Zelenin A.V. Acridine orange as a probe for cell and molecular biology..in: Mason W.T. Fluorescent and Luminescent Probes for Biological Activity. Academic Press, San Diego1999: 117-135Crossref Google Scholar). Acridine orange gives bright green fluorescence bound to DNA in the nucleus and deep red fluorescence bound to RNA in the cytoplasm and nucleoli. When VACV-infected cells were stained with acridine orange, the factories appeared green as expected. However, the DNA staining was not uniform and in some factories there was one large cavity and in others many small ones. Interestingly, these cavities stained red (Figure 1A), suggesting the presence of RNA, since large amounts of single-stranded VACV DNA have not been reported. To further investigate the presence of RNA in virus factories, we employed a technique of introducing antisense oligonucleotide probes into live cells (Carmo-Fonseca et al., 1991Carmo-Fonseca M. Pepperkok R. Sproat B.S. Ansorge W. Swanson M.S. Lamond A.I. In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.EMBO J. 1991; 10: 1863-1873Crossref PubMed Scopus (113) Google Scholar, Politz et al., 1995Politz J.C. Taneja K.L. Singer R.H. Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells.Nucleic Acids Res. 1995; 23: 4946-4953Crossref PubMed Scopus (45) Google Scholar), which was previously used to locate early VACV transcripts in the cytoplasm of infected cells by confocal microscopy (Mallardo et al., 2002Mallardo M. Leithe E. Schleich S. Roos N. Doglio L. Krijnse-Locker J. Relationship between vaccinia virus Intracellular cores, early mRNAs, and DNA replication sites.J. Virol. 2002; 76: 5167-5183Crossref PubMed Scopus (35) Google Scholar, Mallardo et al., 2001Mallardo M. Schleich S. Krijnse-Locker J. Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures.Mol. Biol. Cell. 2001; 12: 3875-3891Crossref PubMed Scopus (49) Google Scholar). Digoxigenin-labeled antisense intermediate stage G8R RNA was transfected in the present study. In uninfected cells, the probe lightly stained the nucleus and appeared as bright aggregates within the cytoplasm (Figure 1B). In infected cells, the antisense probe was concentrated in the factory coincident with multiple areas of low DNA staining (Figure 1C). Serial optical sections showed that cavities containing the probe extended through the entire factory and may be open to the cytoplasm (Movie S1 in the Supplemental Data available with this article online). In factories of some infected cells, the antisense viral RNA probe was predominantly in one large cavity, similar to that seen in the example of acridine orange staining in Figure 1A, or at the factory interface with the cytoplasm (data not shown). However, little or no probe was detected elsewhere in the cytoplasm. In contrast, when infected cells were transfected with a digoxigenin-labeled antisense human glyceraldehyde 3-phosphate dehydrogenase RNA, cytoplasmic staining was seen (data not shown). Nevertheless, we could find some cells in which the probe was associated with factories. The latter association could be due to the presence of RNA-binding proteins in the factories, as will be shown in a following section. Fluorochrome-labeled oligo(dT) was previously used to target poly(A)-containing RNA in live cells (Politz et al., 1995Politz J.C. Taneja K.L. Singer R.H. Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells.Nucleic Acids Res. 1995; 23: 4946-4953Crossref PubMed Scopus (45) Google Scholar). For the same purpose, we transfected biotin-labeled poly(U). In uninfected cells, the poly(U) probe was visualized in the cytoplasm and nucleus (Figure 1D). In infected cells, the probe was also found in the nucleus but was largely absent from the cytoplasm except for the factory region (Figure 1E). Moreover, the factory localization of the poly(U) probe was similar to that of the antisense viral RNA. In contrast, transfected biotin-labeled poly(C) was not found in factories but was detected in the nucleus (data not shown). Taken together, the localization of acridine orange staining and antisense viral RNA and poly(U) probes suggested the presence of mRNA in previously uncharacterized cavities within the viral factories. The apparent absence of polyadenylated mRNA elsewhere in the cytoplasm at 6 hr after infection was consistent with degradation of host messages by this time. E3 is a VACV-encoded double-stranded RNA-binding protein that inhibits the interferon-induced double-stranded RNA-activated protein kinase (PKR) and the 2′, 5′-oligoisoadenylate synthetase-dependent RNase (RNase L) (Chang et al., 1992Chang H.W. Watson J.C. Jacobs B.L. The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase.Proc. Natl. Acad. Sci. USA. 1992; 89: 4825-4829Crossref PubMed Scopus (408) Google Scholar, Rivas et al., 1998Rivas C. Gil J. Melkova Z. Esteban M. Diaz-Guerra M. Vaccinia virus E3L protein is an inhibitor of the interferon (IFN)-induced 2–5A synthetase enzyme.Virology. 1998; 243: 406-414Crossref PubMed Scopus (127) Google Scholar, Yuwen et al., 1993Yuwen H. Cox J.H. Yewdell J.W. Bennink J.R. Moss B. Nuclear localization of a double-stranded RNA-binding protein encoded by the vaccinia virus E3l gene.Virology. 1993; 195: 732-744Crossref PubMed Scopus (117) Google Scholar). E3 is expressed early after infection and at 2 hr had a diffuse cytoplasmic distribution with some nuclear staining (Figure 2A). However, by 4 hr and later times, corresponding to intermediate and late viral gene expression, E3 was largely relocated within and at the surface of the DNA factories (Figure 2B). The redistribution could reflect the location of the large amount of double-stranded viral RNA produced as a consequence of read-through transcription at intermediate and late times (Boone et al., 1979Boone R.F. Parr R.P. Moss B. Intermolecular duplexes formed from polyadenylated vaccinia virus RNA.J. Virol. 1979; 30: 365-374Crossref PubMed Google Scholar). The RNA depots detected in the factories could represent sites of synthesis, accumulation, or degradation. In order to investigate these possibilities, we determined the distribution of the A23 protein subunit of the intermediate transcription factor VITF-3 encoded by VACV (Sanz and Moss, 1998Sanz P. Moss B. A new vaccinia virus intermediate transcription factor.J. Virol. 1998; 72: 6880-6883PubMed Google Scholar). VITF-3 also localized in cavities of the factory as shown in Figure 2C and as serial optical sections in Movie S2. Two cellular proteins, G3BP and Caprin-1 (p137), recently shown to be a heterodimer (Katsafanas and Moss, 2004Katsafanas G.C. Moss B. Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer.J. Biol. Chem. 2004; 279: 52210-52217Crossref PubMed Scopus (61) Google Scholar, Solomon et al., 2007Solomon S. Xu Y. Wang B. David M.D. Schubert P. Kennedy D. Schrader J.W. Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2α, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs.Mol. Cell. Biol. 2007; 27: 2324-2342Crossref PubMed Scopus (155) Google Scholar), are required for in vitro transcription of VACV intermediate genes (Katsafanas and Moss, 2004Katsafanas G.C. Moss B. Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer.J. Biol. Chem. 2004; 279: 52210-52217Crossref PubMed Scopus (61) Google Scholar). These proteins were distributed within the cytoplasm of uninfected cells (Figure 3A) but were relocated to the factory subdomains in infected cells (Figure 3B; Movie S3). Furthermore, G3BP colocalized with poly(A)-containing RNA within the factory (Figure 3C). The viral RNA polymerase was also located in the virus factory but was distributed throughout (data not shown) like virion structural proteins, probably because most of the enzyme is destined for packaging into assembling virus particles. The presence of transcription factors at the same location as the mRNA suggested that the cavities are sites of RNA synthesis and accumulation. The colocalization of cellular G3BP and Caprin-1 (p137) with viral mRNA was consistent with previous evidence for a role in VACV transcription. Nevertheless, it raised the important question of whether cellular proteins might relocate to these subdomains nonspecifically. We therefore determined the effect of VACV infection on several different proteins including NF-κB and CRKL. The major form of NF-κB resides in the cytoplasm in an inactive state and is activated and transported to the nucleus in response to a variety of stimuli (Baeuerle and Baltimore, 1988Baeuerle P.A. Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor.Cell. 1988; 53: 211-217Abstract Full Text PDF PubMed Scopus (779) Google Scholar). CRKL is an adaptor protein that can translocate to different regions of the cell (ten Hoeve et al., 1993ten Hoeve J. Morris C. Heisterkamp N. Groffen J. Isolation and chromosomal localization of CRKL, a human crk-like gene.Oncogene. 1993; 8: 2469-2474PubMed Google Scholar). Following VACV infection, NF-κB retained a cytoplasmic distribution, and CRKL was distributed throughout the cell (Figure S1). Thus, the presence of certain viral and cellular proteins within the cavities seems specific. The presence of viral mRNA exclusively within the factory suggested that it is translated nearby. Indeed, we found that cellular translation initiation factors eIF4G (Figure 4A) and eIF4E (Figure 4B) were relocated from the cytoplasm to the factories where they colocalized with G3BP in the cavities. Serial optical sections demonstrating the factory distribution of eIF4E are shown in Movie S4. Although antibody to the large ribosomal subunit P proteins stained the factories exclusively in the cavities, there was similar intensity staining of the cytoplasm outside of the factories (data not shown). These data indicated that a smaller proportion of the ribosomes relocated to factories compared to the translation factors. The intracellular redistribution of translation factors led us to consider that viral protein synthesis might occur within the factory rather than in the surrounding cytoplasm. To investigate this hypothesis, we determined the location of newly synthesized proteins encoded by the viral genome. A recombinant VACV with the Escherichia coli lacZ gene regulated by a VACV intermediate promoter was chosen, as it seemed unlikely that β-galactosidase would be targeted to the virus factory if it were synthesized outside of it. Remarkably, at 6 hr after infection, β-galactosidase was predominantly in the factory (Figures 5A and 5B). Later, as the factories became less discrete, the protein was more diffusely distributed in the cytoplasm (data not shown). Although the above experiment strongly suggested the coordination of viral transcription and translation within the factory, we devised a more decisive experiment. Previous studies indicated that the number of replication sites in a cell is related to the number of virus particles used for infection (Cairns, 1960Cairns J. The initiation of vaccinia infection.Virology. 1960; 11: 603-623Crossref PubMed Scopus (99) Google Scholar, Mallardo et al., 2002Mallardo M. Leithe E. Schleich S. Roos N. Doglio L. Krijnse-Locker J. Relationship between vaccinia virus Intracellular cores, early mRNAs, and DNA replication sites.J. Virol. 2002; 76: 5167-5183Crossref PubMed Scopus (35) Google Scholar). Those data suggested that a virus factory might arise from a single virus particle. We devised a way of demonstrating this and testing our hypothesis regarding the colocalization of transcription and translation. Two recombinant VACVs were constructed: one expressed the A5 core protein (Carter et al., 2003Carter G.C. Rodger G. Murphy B.J. Law M. Krauss O. Hollinshead M. Smith G.L. Vaccinia virus cores are transported on microtubules.J. Gen. Virol. 2003; 84: 2443-2458Crossref PubMed Scopus (83) Google Scholar) fused to yellow fluorescent protein (YFP), and the other expressed A5 fused to cyan fluorescent protein (CFP). The two fluorescent proteins differ by only a few amino acids, so that the recombinant viruses were nearly identical though easily distinguishable by confocal fluorescent microscopy. Since A5 is a core protein (Maa and Esteban, 1987Maa J.-S. Esteban M. Structural and functional studies of a 39,000-Mr immunodominant protein of vaccinia virus.J. Virol. 1987; 61: 3910-3919Crossref PubMed Google Scholar), it must be present within the virus factory for assembly to occur. This localization could result from synthesis within the factory or synthesis outside the factory followed by targeting to assembly sites. We considered that, if a cell was infected with one of each recombinant virus particle and generated two factories, one should exhibit yellow fluorescence and the other should exhibit cyan fluorescence provided that each factory was derived from a single genome and translation occurred within the factory. On the other hand, if mRNAs were exported from the factory prior to translation, the factories should contain a mixture of YFP and CFP. We infected cells with a 1:1 mixture of the two viruses and looked for individual cells that exhibited both yellow and cyan fluorescence. Among those cells were many examples with individual yellow and cyan factories (Figures 5C–5E). In addition, there were factories that exhibited both cyan and yellow fluorescence (Figure 5F), suggesting an origin from two or more particles. Even in the latter, however, there were distinct yellow and blue granules that presumably represent individual or aggregated virions containing multiple copies of either A5-YFP or A5-CFP. Prokaryotes coordinate gene expression by coupling transcription and translation (French et al., 2007French S.L. Santangelo T.J. Beyer A.L. Reeve J.N. Transcription and translation are coupled in Archaea.Mol. Biol. Evol. 2007; 24: 893-895Crossref PubMed Scopus (54) Google Scholar, Miller et al., 1970Miller Jr., O.L. Hamkalo B.A. Thomas Jr., C.A. Visualization of bacterial genes in action.Science. 1970; 169: 392-395Crossref PubMed Scopus (221) Google Scholar). In eukaryotes, however, the nuclear membrane separates replication and transcription from translation, a functional division that is maintained following infection by most DNA viruses. Poxviruses are exceptional, however, because they replicate within the cytoplasm in foci called DNA factories that have been likened to mini-nuclei (Schramm and Locker, 2005Schramm B. Locker J.K. Cytoplasmic organization of poxvirus DNA replication.Traffic. 2005; 6: 839-846Crossref PubMed Scopus (65) Google Scholar). Here we showed that the latter term is not appropriate at late times, when transcription and translation occur within the poxvirus DNA factory. In addition to enhancing VACV replication efficiency, colocalization of these two processes may give poxviruses a competitive advantage, contributing to the takeover of cell functions. The transcription of VACV early genes occurs within cytoplasmic core particles, and the mRNAs are transported via microtubules to distant sites (Mallardo et al., 2001Mallardo M. Schleich S. Krijnse-Locker J. Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures.Mol. Biol. Cell. 2001; 12: 3875-3891Crossref PubMed Scopus (49) Google Scholar). Although DNA replication and virion assembly were known to occur within poxvirus DNA factories, the location of intermediate and late viral mRNA had not previously been determined. We found that RNA was localized within cavities or tunnels, denoted by diminished DNA staining, and on the surface of the factory. Optical sections and accessibility of a transfected antisense probe suggested that the cavities connect with the cytoplasm and in that way increase the factory/cytoplasm interface area. The viral E3 double-stranded RNA-binding protein, which prevents activation of PKR and RNase L innate immune defenses (Chang et al., 1992Chang H.W. Watson J.C. Jacobs B.L. The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase.Proc. Natl. Acad. Sci. USA. 1992; 89: 4825-4829Crossref PubMed Scopus (408) Google Scholar, Rivas et al., 1998Rivas C. Gil J. Melkova Z. Esteban M. Diaz-Guerra M. Vaccinia virus E3L protein is an inhibitor of the interferon (IFN)-induced 2–5A synthetase enzyme.Virology. 1998; 243: 406-414Crossref PubMed Scopus (127) Google Scholar), was also present in the cavities. The additional presence of the virus-encoded intermediate transcription factor suggested that the cavities were sites of RNA synthesis and not merely storage depots. Finding the G3BP/Caprin-1 (p137) heterodimer, which we previously showed to complement intermediate transcription in vitro (Katsafanas and Moss, 2004Katsafanas G.C. Moss B. Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer.J. Biol. Chem. 2004; 279: 52210-52217Crossref PubMed Scopus (61) Google Scholar), concentrated in the cavities provided the first in vivo evidence for a role of this host protein in viral transcription. In contrast, cellular NF-κB and CRKL, cytoplasmic regulatory proteins that have not been implicated in VACV transcription, were not concentrated in the factory. In addition, our localization of viral and cellular RNA-binding proteins and transcription factors in RNA subdomains of the factory contrasts with the partial leakage of some nuclear proteins (e.g., SP1, TBP, and YY1) that diffusely localize to VACV replications sites evidently due to their DNA-binding domains (Oh and Broyles, 2005Oh J. Broyles S.S. Host cell nuclear proteins are recruited to cytoplasmic vaccinia virus replication complexes.J. Virol. 2005; 79: 12852-12860Crossref PubMed Scopus (32) Google Scholar). It will be interesting to determine the intracellular localization of hnRNP A2 and RBM3, RNA-binding proteins that have been reported to stimulate late transcription in vitro (Wright et al., 2001Wright C.F. Oswald B.W. Dellis S. Vaccinia virus late transcription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins.J. Biol. Chem. 2001; 276: 40680-40686Crossref PubMed Scopus (52) Google Scholar). The recruitment of mRNA to the ribosome is controlled by the activity of the translation factor eIF4E, the component of eIF4F that binds to the 5′-cap structure of mRNA (Kapp and Lorsch, 2004Kapp L.D. Lorsch J.R. The molecular mechanics of eukaryotic translation.Annu. Rev. Biochem. 2004; 73: 657-704Crossref PubMed Scopus (384) Google Scholar). Translation initiation is dependent upon the interaction of eIF4E with the scaffold protein eIF4G and other factors. Therefore, the finding that eIF4E and eIF4G were concentrated in the cavities suggested that poxvirus transcription and translation are linked, though probably not directly coupled as in bacteria. The presence of ribosomes in the cavities was suggested by staining with antibody to the P proteins of the large ribosomal subunit. However, ribosomes were still distributed throughout the cytoplasm outside of factory areas. In most situations, translation initiation factors such as eIF4E, rather than ribosomes, are limiting for protein synthesis (Hershey, 1991Hershey J.W. Translational control in mammalian cells.Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (837) Google Scholar). The RNA-containing subdomains within poxvirus factories share some features with cytoplasmic RNA granules present in the cytoplasm of uninfected cells (Anderson and Kedersha, 2006Anderson P. Kedersha N. RNA granules.J. Cell Biol. 2006; 172: 803-838Crossref PubMed Scopus (802) Google Scholar). Thus, both cellular stress granules and the factory subdomains contain mRNA, eIF4E, eIF4G, and G3BP. However, the stress granules are thought to regulate cellular mRNA translation, whereas viral RNA appears to be synthesized and translated in association with the factory subdomains. Although the presence of translation initiation factors and ribosomal proteins provided circumstantial evidence that protein synthesis occurs in the factory, more compelling data were needed. First, we infected cells with a recombinant VACV expressing the β-galactosidase reporter gene and found that the protein was located in the factory despite the improbability of specific targeting signals. Evidently, the matrix of the factory prevented the rapid diffusion of β-galactosidase from its site of synthesis into the surrounding cytoplasm. Even more compelling evidence that viral proteins are synthesized in the factory was obtained by infecting cells simultaneously with two recombinant viruses, one expressing a VACV core protein fused to CFP and the other expressing the same VACV core protein fused to YFP. The finding of individual yellow and cyan factories in the same cell could be explained if a factory formed from a single virus particle and both transcription and translation occurred within the factory boundary. This result would be difficult to explain if viral mRNAs were translated in the surrounding cytoplasm, where they would be likely to mix. Not surprisingly, we also found factories containing both CFP and YFP. In some cases yellow and cyan sectoring indicated that the two factories had fused, while in others there was mixing of yellow and cyan, suggesting that a factory had formed from two particles. The latter could arise from aggregated particles, cores that traffic along microtubules to a similar juxtanuclear destination (Carter et al., 2003Carter G.C. Rodger G. Murphy B.J. Law M. Krauss O. Hollinshead M. Smith G.L. Vaccinia virus cores are transported on microtubules.J. Gen. Virol. 2003; 84: 2443-2458Crossref PubMed Scopus (83) Google Scholar), or the merging of replication sites. The synthesis of viral RNA and proteins within the factory in which genome replication and virion assembly occur provides obvious production efficiency. However, there may be an additional benefit. Poxviruses encode a variety of proteins that limit specific host responses at early times of infection. At later stages of infection, there is a global inhibition of host gene expression. How the latter occurs has been subject to much speculation over the years (Moss, 2007Moss B. Poxviridae: The viruses and their replicaton.in: Knipe D.M. Fields Virology. Lippincott Williams & Wilkins, Philadelphia2007: 2905-2946Google Scholar). One mechanism is mRNA destabilization, a process that is initiated by a decapping enzyme that is encoded by all poxviruses (Parrish et al., 2007Parrish S. Resch W. Moss B. Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression.Proc. Natl. Acad. Sci. USA. 2007; 104: 2139-2144Crossref PubMed Scopus (80) Google Scholar). The present study provides evidence for a second mechanism enabled by the sequestering of crucial translation initiation factors within cytoplasmic DNA factories, where viral transcription and translation occur. Uninfected or infected (1 PFU/cell) HeLa cells on 12 mm round glass coverslips were washed (3× for 5 min) in Dulbecco's phosphate-buffered saline, fixed in 4% paraformaldehyde for 20 min, washed again, permeabilized with 0.1% Triton X-100 in PBS for 15 min, washed again, and incubated at room temperature for 1 hr in phosphate-buffered saline with 1% bovine serum albumin. Primary antibodies were diluted 1:100 in phosphate-buffered saline with 1% bovine serum albumin and incubated at room temperature for 6 hr or overnight at 4°C. After washing, secondary antibodies were applied at a 1:100 dilution as above. When cells were dual labeled the procedure was repeated. Nuclei and factories were stained with DAPI at a concentration of 5 μg/ml in H2O at room temperature for 15 min and washed. Coverslips were mounted on 75 × 25 mm slides using Mowiol as the mounting medium. Cells prepared as above were stained with 6 μg/ml of acridine orange (Molecular Probes, Eugene, OR), washed, and mounted on glass slides. The metachromatic dye was excited at 488 nm and detector slits were set between 510 and 549 nm and 626 and 656 nm to visualize double- and single-stranded nucleic acids, respectively. Images were collected on a Leica SP2 AOBS confocal microscope (Leica Microsystems, Bannockburn, IL) using a 63× oil immersion objective NA 1.32 or 1.4 zoom X. Fluorochromes were excited using 357 or 405 nm for DAPI, 488 nm for Alexa Fluor 488, 458 for CFP, 514 nm for YFP, and 594 nm excitation for Alexa Fluor 594. Detector slits were configured to minimize crosstalk between the channels. Images were collected sequentially in a z series, deconvolved using Huygens 2.10 (Scientific Volume Imaging, Hilversum, The Netherlands) and processed using Imaris 5.5.1 (Bitplane AG, Zurich, Switzerland) and Adobe Photoshop CS (Adobe Systems, San Jose, CA). The G8R gene was amplified by PCR using an N-terminal primer (5′-GCGCGTCGACATGAGCATCCGTATAAAAATCGATAAACTGCGCC-3′) and a C-terminal primer (5′-AATTGCATGCTAATACGACTCACTATAGGGTTAATCTAAAAACGCCATAAAGATGTTGATCTTAAAGG-3′), which included the sequence of the bacteriophage T7 RNA polymerase promoter. The 785 nucleotide product was purified with a QIAquick PCR purification kit (QIAGEN, Fremont, CA), and 1 μg was mixed with 2 μl of 10× DIG labeling mix (10 mM ATP, 10 mM CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM digoxigenin-11-UTP [pH 7.5]), 2 μl of 10× transcription buffer (0.4 M Tris-HCl [pH 8.0], 60 mM MgCl2, 0.1 M dithiotreitol, 20 mM spermidine), 2 μl of T7 RNA polymerase in a total volume of 20 μl (reagents from Roche Applied Science, Indianapolis, IN). This mixture was incubated for 2 hr at 37°C, and the reaction was stopped with 2 μl of 0.2 M EDTA (pH 8.0). The transcript was shown to be pure and of the correct size by electrophoresis of 1 μl on a 6% Tris borate EDTA urea gel and staining with ethidium bromide. Nucleic acid concentration was determined by absorbance, and 350 ng mixed with lipofectamine (Invitrogen, Carlsband, CA) was used to transfect uninfected or infected HeLa cells that had been plated on 12 mm round coverslips. For localization of human glyceraldehyde 3-phosphate dehydrogenase, in vitro transcriptions were carried out as above using the linearized pTRI-GAPDH-plasmid, which includes the sequence of the bacteriophage T7 RNA polymerase promoter (Ambion, Austin, TX). Absorbance was determined, and 350 ng of the 387 nucleotide product was used to transfect uninfected or infected HeLa cells that had been plated on 12 mm round coverslips. To localize polyadenylated mRNAs, a 5′ biotinylated 40-mer poly(U) was chemically synthesized (Dharmacon, Lafayette, CO), and 1 μg was used for transfection as described for the antisense G8R RNA. Open reading frames encoding CFP and YFP were amplified using plasmid templates from Clonetech (Mountain View, CA). Fusion of the open reading frames to the N-terminal codon of A5L was achieved by overlap extension PCR using VACV WR genomic DNA as the template. One microgram of each PCR product was transfected into HeLa cells that had been infected with VACV vRPO22C10H (Katsafanas and Moss, 1999Katsafanas G.C. Moss B. Histidine codons appended to the gene encoding the RP022 subunit of vaccinia virus RNA polymerase facilitate the isolation and purification of functional enzyme and associated proteins from virus-infected cells.Virology. 1999; 258: 469-479Crossref PubMed Scopus (9) Google Scholar). The cell lysate of each separate transfection/infection was plated onto BS-C-1 cells, and plaques were picked by their ability to fluoresce. Virus in cyan and yellow plaques were each clonally purified four times on BS-C-1 cells. The recombinant viruses were amplified in HeLa cells to achieve high-titer virus stocks. Antibodies and their sources were as follows: anti-G3BP mouse mAb (BD PharMingen, San Diego, CA); rabbit polyclonal anti-G3BP and rabbit polyclonal anti-Caprin-1 (p137) (Katsafanas and Moss, 2004Katsafanas G.C. Moss B. Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer.J. Biol. Chem. 2004; 279: 52210-52217Crossref PubMed Scopus (61) Google Scholar); mouse mAb anti-E3 (Yuwen et al., 1993Yuwen H. Cox J.H. Yewdell J.W. Bennink J.R. Moss B. Nuclear localization of a double-stranded RNA-binding protein encoded by the vaccinia virus E3l gene.Virology. 1993; 195: 732-744Crossref PubMed Scopus (117) Google Scholar); rabbit polyclonal anti-A23 (unpublished data); rabbit polyclonal anti-β galactosidase (Abcam, Inc, Cambridge, MA); goat polyclonal anti-eIF4G, rabbit polyclonal anti-NF-κB and CRKL, mouse mAb anti-eIF4E (Santa Cruz Biotechnology, Santa Cruz, CA); sheep anti-digoxigenin-fluorescein (Roche Applied Sciences); Alexa Fluor 488 and Alexa Fluor 594 conjugated to anti-IgG of appropriate species (Molecular Probes). Alexa Fluor 488 conjugated to streptavidin and anti-Alexa Fluor 488 rabbit IgG were from Molecular Probes. We thank Owen Schwartz and Juraj Kabat for training with confocal microscopy and imaging software and Ted Pierson for comments on the manuscript. The study was supported by the Division of Intramural Research, NIAID, NIH. Download .mov (.43 MB) Help with mov files Movie S1. Movie of Optical Sections through Factory Showing Antisense Viral RNA at 6 hr after Infection with VACVHeLa cells were infected with VACV and transfected 1 hr later with antisense G8R RNA labeled with digoxigenin UTP. At 6 hr after infection, the cells were washed, fixed, and stained with sheep anti-digoxigenin-fluorescein, followed by Alexa Fluor 488 donkey anti-sheep (green) and DAPI (blue). Optical sections correspond to Figure 1C. Download .mov (.87 MB) Help with mov files Movie S2. Movie of Optical Sections through a Factory Showing Distribution of the Intermediate Transcription FactorVACV-infected HeLa cells were fixed after 8 hr, processed and stained with rabbit polyclonal anti-A23, followed by Alexa Fluor 488 goat anti-rabbit IgG (red) and DAPI (blue). Corresponds to Figure 2C. Download .mov (.78 MB) Help with mov files Movie S3. Movie of Optical Sections through a Factory Showing Distribution of G3BP and Caprin-1 (p137)HeLa cells infected with VACV were processed after 8 hr and sequentially stained with mouse MAb anti-G3BP (green), rabbit polyclonal anti-p137 (red), Alexa Fluor 488 goat anti-mouse IgG, and Alexa Fluor 594 goat anti-rabbit IgG and DAPI. Optical sections of the composite are shown. Corresponds to Figure 3B. Download .mov (1.29 MB) Help with mov files Movie S4. Movie of Optical Sections of a Factory Showing the Distribution of eIF4EAt 8 hr after VACV infection, HeLa cells were stained sequentially with mouse MAb to eIF4E (green), rabbit polyclonal anti-G3BP (red), Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 594 goat anti-rabbit IgG, and DAPI. DNA and eiF4E staining are shown in the optical sections. Corresponds to Figure 4A. Download .pdf (.66 MB) Help with pdf files Figure S1. Distribution of NF-κB and Crk-L in VACV-Infected HeLa Cells(A) HeLa cells were fixed at 6 hr after VACV infection and stained with rabbit polyclonal anti-NF-κB (p65) followed by Alexa Fluor 594 goat anti-rabbit (red), and DAPI (blue).(B) HeLa cells were fixed at 8 hr after VACV infection and stained with rabbit polyclonal anti-Crk-L, Alexa Fluor 488 goat anti-rabbit IgG (green), and DAPI (blue)." @default.
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• W2003702834 title "Colocalization of Transcription and Translation within Cytoplasmic Poxvirus Factories Coordinates Viral Expression and Subjugates Host Functions" @default.
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